The long-term goal of this proposal is to identify mitochondrial proteins that facilitate the transport of heme intermediates into and within the mitochondria and to outline the physiological processes that require the function of specific transporters of heme synthesis intermediates. I have previously shown that Tmem14c is required for terminal erythropoiesis and import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis. Tmem14c deficiency causes anemia and porphyrin accumulation in our genetic models. However, several aspects of TMEM14C function and protoporphyrinogen IX transport are still unclear and lead to two main hypotheses that are addressed in my Specific Aims. Firstly, the mechanism by which TMEM14C facilitates the transport of protoporphyrinogen IX is still not understood. As the tight structure of TMEM14C suggests that it functions as a transmembrane channel, I hypothesize that TMEM14C directly transports protoporphyrinogen IX into the mitochondrial matrix. I will test this in Specific Aim 1 by quantifying the relative affinities of TMEM14C to heme and tetrapyrrolic heme intermediates. I will also measure the rates of in vitro heme synthesis in wild-type and Tmem14c deficient mitochondria in the presence of exogenous heme intermediates. Secondly, erythroid cells lacking TMEM14C have survival rates, mitochondrial potentials and mitochondrial masses similar to wild-type cells. As mitochondrial and cellular respiration, which are essential life-sustaining processes require hemoproteins (proteins with heme-cofactors), it is probable that cells possess other protoporphyrinogen IX transporters that maintain housekeeping heme synthesis. As the structures of TMEM14 proteins are very similar, I hypothesize that other members of the TMEM14 superfamily function as protoporphyrinogen IX transporters to maintain housekeeping heme synthesis and cellular physiology. In Specific Aim 2, I will test this hypothesis by knocking down TMEM14 genes in vertebrate cell lines and quantifying the effects of the knockdown on heme synthesis. Candidate TMEM14 genes involved in heme synthesis will be knocked down in primary hepatocytes and primary hematopoietic cells to examine their effect on mitochondrial physiology and hemoglobinization. I will test the in vivo requirement for tmem14 genes in erythroid and hepatic development by knockdown studies in the zebrafish. The completion of my project will shed light on the genetics and biochemistry of the heme synthesis pathway and will contribute to our fundamental understanding of the pathological consequences that occur when the pathway is perturbed by disruptions to the transport of heme intermediates. The specific aims and career development plan described in this proposal are a logical continuation of my prior training but will provide a framework by which I will scientifically and differentiate myself from my advisors, ultimately paving the way for a successful transition to independence.